Field of the Invention
The present invention relates to hepatitis B virus (hereinafter it refers to HBV) polymerase containing a histidine tag, RNase H enzyme derived from HBV polymerase and processes for preparation thereof.
More particularly, the present invention relates to recombinant HBV polymerase, its RNase H domain with enzyme activity, expression vectors producing the enzymes in E. coli and processes for preparing the HBV polymerase and the RNase H enzyme which can be easily purified due to their histidine tags.
And the present invention relates to uses of the HBV polymerase and the RNase H enzyme for screening antiviral agents.
HBV is the main virus among hepatitis viruses, which infects more than 300 million people worldwide. HBV causes acute or chronic hepatitis, which results in liver cirrhosis or liver cancer (Tiollais and Buenda, Scientific American, 264: 48-54, 1991 ; Blumberg, B. S., Background and perspective in advances in hepatitis research, F. V. Chisari, ed., New York, Mason publishing, 1984). Because of molecular characteristics of HBV and its close relation with liver diseases, various researches about HBV have been accomplished.
HBV is a DNA virus, a member of the hepadnaviridae family, which has a spherical structure composed of nucleocapsid and core. HBV genome is a partially double stranded DNA of only 3.2 kb size, which is not a circular form. In detail, HBV genome is composed of four overlapped genes that are the polymerase (P) gene, the surface protein (HBsAg; S, pre-S1, pre-S2) gene, the core protein (HBcAg; pre-C, C) gene and X protein (HBx) gene. Among these genes, X protein gene encodes regulatory protein, and the other genes encode structural proteins of HBV. The polymerase gene occupies 80% of the total genome and encodes 94 KD-sized protein composed of 845 amino acids.
HBV infects hepatic cells by the process described below. The specific receptor of the hepatic cell recognizes the surface protein on the surface of the virion particle and binds with them so as to draw the virion into the hepatic cell. Then HBV polymerase synthesizes the single-stranded part of partially double-stranded DNA in order to obtain complete HBV genome. And the HBV genome of 3.2 kb size is transcribed with cellular RNA polymerase to produce pre-genomic mRNA of about 3.5 kb, core protein (C) mRNA, surface protein mRNA and X protein mRNA. Viral proteins are translated from these mRNAs. Specially HBV polymerase synthesizes an RNA intermediate with its reverse transcriptase activity so as to provide a template for the DNA genome and make a replicasome structure with the pre-genomic mRNA, the core protein and the like, which is called encapsidation process. The HBV genome can be encapsidated easily since 3'-terminus of the polymerase containing continuous glutamic acid residues has affinity with nucleic acids. The above RNA intermediate in the replicasome serves as a template for minus strand DNA synthesis and then the full-length minus strand serves as a template for plus strand DNA synthesis by DNA-dependent DNA polymerase (DDDP) activity of the polymerase so as to make total pre-genomic mRNAs finally. By repeating the above process, more than 200-300 copies of the genomic DNA is maintained in pool and the viral proteins mentioned above are expressed (Tiollais and Buenda, Scientific American, 264: 48-54, 1991 ; Ganem, D. and Varmus, H. E., Annu. Rev. Biochem., 56: 651-693, 1987).
Interestingly, HBV replicates its genome by using the RNA intermediate and reverse transcription even though it is a DNA virus. It is-known that retrovirus exploits the reverse transcription to replicate its genome. Particularly, the polymerase of retrovirus is reported to be a multifunctional enzyme which shows DNA-dependent DNA polymerase activity, reverse transcriptase activity, and RNase H activity. It is remarkable that HBV polymerase contains a series of functions necessary for the replication of virus genome. Namely, the following functions: (i) protein primer, (ii) RNA-dependent DNA polymerase (RT), (iii) DNA-dependent DNA polymerase (DDDP), (iv) RNase H activity consist in one polypeptide. The reverse transcriptase activity of HBV polymerase was first reported by Kaplan et al., and has been exploited to elucidate the mecahnism of HBV replication.
As mentioned above, a reverse transcriptase has an active RNase H domain commonly which recognizes RNA/DNA complex and hydrolyzes only the RNA strand selectively. The RNase H activity is indispensable to the reverse transcription, since the reverse transcriptase can replicate DNA continuously only after RNA intermediate is hydrolyzed by the RNase H activity. Although RNase H enzyme is known as a domain of the reverse transcriptase recently, RNase H enzyme was first discovered in the calf thymus by Hausen and Stein, and has been reported from various prokaryotes and eukaryotes (Stein, Hans and Hausen, P., Science, 166 393-395, 1969).
Generally speaking, an active RNase H domain of HBV polymerase is localized within its C-terminus. The amino acid sequence and nucleotide sequence of the polymerase were reported to be very similar to those of the polymerase of Moloney murine Leukemia Virus. In addition, an active RNase H domain of HBV polymerase was known to synthesize a plus strand primer which can be derived from the pre-genomic RNA putatively. Particularly, it was identified by performing mutagenesis that the conserved sequence in the RNase H enzyme was necessary for viral proliferation. In addition, the RNase H domain plays a role to synthesize a minus strand DNA as well as the plus strand DNA and to perform RNA packaging, which is identified by mutating the RNase H domain of duck HBV polymerase. But it is reported recently that duck HBV polymerase can not recognize binding region E within the pre-genomic RNA of human HBV.
Therefore, human HBV polymerase and its RNase H domain should be studied directly in addition to indirect researches by utilizing duck HBV polymerase in order to elucidate human HBV and the mechanism of its polymerase. Hitherto, the surface protein and the X protein which is necessary for the development of vaccines and for the regulation of transcription in proceeding liver cancer respectively has been studied actively. However, HBV polymerase has seldom been exploited although it can be used to develop antiviral agents. Because HBV polymerase is difficult to be separated from virus particle and to obtain sufficient amounts, especially as an active form (Radziwill, G. et al., Virology, 163: 123-132, 1988). Presently, in order to develop novel therapeutical agents for hepatitis, cell lines infected with HBV have been used for screening antiviral agents. However, effective therapeutical agents has not been yet developed, since it takes longer time and costs more for a screening method using cell lines than for screening methods using HBV polymerase or its RNase H enzyme.
Recently in order to elucidate HBV, HBV polymerase and its RNase H domain have been studied as descibed above. Particularly researches for the mass production of above enzymes have been attempted by using recombinant DNA technology. The inventors of the present invention have produced a recombinant HBV polymerase which is expressed from E. coli transformant, measured its enzyme activity and filed a patent application thereof (Korean Patent Application 94-3918). The recombinant HBV polymerase was produced in E. coli as a form of fusion protein with maltose binding protein (MBP), and can be easily purified by MBP affinity column chromatography. But active HBV polymerase is difficult to be obtained massively because the polymerase can be degradaded at the C-terminus and has low purity.
Foreign proteins can be obtained massively by inserting a histidine tag into the proteins by recombinant DNA technology. The nucleotide sequences encoding histidine tag is inserted into the 5'-terminus or 3'-terminus of the gene, and the histidine-tag prevents degradation of the recombinant protein so as to prepare stable enzyme. In addition, the highly active recombinant protein can be purified easily by using histidine tag affinity column as a metal chelating affinity column.
In order to develop effective therapeutical agents, HBV polymerase and its RNase H enzyme have been produced by processes of the present invention. The inventors constructed expression vectors containing HBV polymerase gene with nucleotide sequences encoding a histidine tag at the C-terminus of the recombinant protein and expression vectors containing RNase H domain gene which is derived from the 3'-terminus of the HBV polymerase gene. In addition, HBV polymerase and its RNase H domain have been produced as forms of fusion protein massively in E. coli by using the expression vectors and purified easily by using amylose column and histidine tag affinity column. Thus highly active and stable HBV polymerase and its RNase H enzyme which are not degradaded can be prepared. Furthermore, the inventors have developed novel screening methods for antiviral agents by using the HBV polymerase and its RNase H domain of the present invention.